A fragmentation geometry based upon axial acceleration of m/z-selected protein ions into a linear octopole ion trap allowed simultaneous production and external accumulation of fragment ions prior to m/z measurement in a FT mass spectrometer. Improved dynamic range resulting from this octopole collisionally activated dissociation resulted in a 2.5؋ increase in experimental throughput and a 2؋ increase in fragment ion matches to gene products identified and characterized in the top down fashion. The acceleration voltage for optimal fragmentation has a m/z and mass dependence, knowledge of which facilitated an automated platform for top down MS/MS on a quadrupole FT hybrid mass spectrometer. Controlled by improved software for data acquisition (e.g. using dynamic exclusion of previously identified species), automated octopole collisionally activated dissociation of samples fractionated using chromatofocusing and reversed-phase liquid chromatography achieved a significant increase in protein identification rate versus previous benchmarks. Also a batch analysis version of ProSight PTM facilitated probability-based identification of intact proteins obtained in a higher throughput fashion. In total, 101 unique proteins (5-59 kDa) were identified from whole cell lysates of Methanosarcina acetivorans grown anaerobically, including the characterization of several mispredicted start sites and biologically relevant mass discrepancies. Molecular & Cellular Proteomics 5:14 -25, 2006.Over the last 3 years a platform consisting of two-dimensional fractionation (1), an automated (2, 3) quadrupole (Q) 1 FT mass spectrometer (4), and new informatic tools (5-7) has emerged for "top down" analysis (8 -11) of intact proteins with DNA-predicted sequences. Application of the nascent platform to Methanococcus jannaschii and Saccharomyces cerevisiae led to the identification of ϳ230 expressed gene products (Ͻ39 kDa) with 100% sequence coverage and simultaneous characterization of ϳ50 post-translational modifications (PTMs) (12, 13). Although yielding promising results, throughput has been limited compared with bottom up platforms based on tryptic peptides (14 -19).Poor dynamic range associated with ESI (20) of intact proteins in complex mixtures, with up to 15 observed proteins per fraction, is minimized by preselection of pseudomolecular ions with quadrupole mass filter-FT or quadrupole ion trap-FT hybrid instruments (4,(21)(22)(23)(24)(25). Given limitations in ion transmission of the quadrupole mass filter at 1-Da resolution, isolation windows in excess of ⌬m/z ϭ 5 were commonly used in prior studies for multiplexed fragmentation of several precursor proteins at once and detection of hundreds of fragment ions (4). This challenges the specificity of protein identification because the search depends largely upon the mass accuracy and the ratio n/f of the number of matching fragment ions for a protein in a database (n) relative to the number observed (f) (5).Controlled fragmentation with infrared multiphoton dissociation (26) facilitate...

Characterizing combinations of coding polymorphisms (cSNPs), alternative splicing and posttranslational modifications (PTMs) on a single protein by standard peptide-based proteomics is challenging owing to <100% sequence coverage and the uncoupling effect of proteolysis on such variations >10-20 residues apart. Because top down MS measures the whole protein, combinations of all the variations affecting primary sequence can be detected as they occur in combination. The protein form generated by all types of variation is here termed the "proteotype", akin to a haplotype at the DNA level. Analysis of proteins from human primary leukocytes harvested from leukoreduction filters using a dual on-line/off-line top down MS strategy produced >600 unique intact masses, 133 of which were identified from 67 unique genes. Utilizing a two-dimensional platform, termed multidimensional protein characterization by automated top down (MudCAT), 108 of the above protein forms were subsequently identified in the absence of MS/MS in 4 days. Additionally, MudCAT enables the quantitation of allele ratios for heterozygotes and PTM occupancies for phosphorylated species. The diversity of the human proteome is embodied in the fact that 32 of the identified proteins harbored cSNPs, PTMs, or were detected as proteolysis products. Among the information were three partially phosphorylated proteins and three proteins heterozygous at known cSNP loci, with evidence for non-1:1 expression ratios obtained for different alleles.Mass spectrometry-based proteomics is now a crucial component of modern structural biology. 1 The next phase of maturation for interrogation of proteomes involves more than a description of events, but how these diverse events occur in combination, thereby requiring a portion of the proteomic discussion to move from its "descriptive" beginnings (e.g., cataloging expressed proteins and site-specific modifications, etc.) into an integrative, combinatorial mode. Within the human proteome, much discussion is focused on modifications, splice variants, and polymorphisms/mutations; all the combinations of which serve to cloud the biology behind their presence. 2,3 Integration of all such complexity into an array of protein forms from a single gene, "the proteotype", produces molecular information akin to genetecists' haplotypes. Categorizing the proteotype of a given gene product requires sophisticated mass spectrometric techniques to streamline detection of combinations of coding polymorphisms (cSNPs) and post-translational modifications (PTMs).Application of established technology to clinically significant groups of samples (clinical proteomics) has ushered into existence the notion of "population proteomics", 4 wherein biomarkers at the protein level are detected and identified for further focus in drug target discovery and clinical diagnostics (e.g., ELISA-based screening). 5 The majority of population *To whom correspondence should be addressed. E-mail: Kelleher@scs.uiuc Recent publications report diverse methods for pu...

For automated production of tandem mass spectrometric data for proteins and peptides >3 kDa at >50 000 resolution, a dual online-offline approach is presented here that improves upon standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies. An integrated hardware and software infrastructure analyzes online LC-MS data and intelligently determines which targets to interrogate offline using a posteriori knowledge such as prior observation, identification, and degree of characterization. This platform represents a way to implement accurate mass inclusion and exclusion lists in the context of a proteome project, automating collection of highresolution MS/MS data that cannot currently be acquired on a chromatographic time scale at equivalent spectral quality. For intact proteins from an acid extract of human nuclei fractionated by reversed-phase liquid chromatography (RPLC), the automated offline system generated 57 successful identifications of protein forms arising from 30 distinct genes, a substantial improvement over online LC-MS/MS using the same 12 T LTQ FT Ultra instrument. Analysis of human nuclei subjected to a shotgun Lys-C digest using the same RPLC/automated offline sampling identified 147 unique peptides containing 29 co-and post-translational modifications. Expectation values ranged from 10 −5 to 10 −99 , allowing routine multiplexed identifications.Historically, there has been a trade-off in mass spectrometry between resolution and sensitivity. In contemporary MS-based proteomics, 1 there is long-standing interest in increasing either spectral resolution, the size of peptides/proteins analyzed, or both. Such improvements will allow more protein complexity to be measured with greater certainty. 2 Driven in part by new ion fragmentation approaches 3,4 and improving instrumentation, 5-12 the steadily advancing capabilities of MS are challenged by targeting polypeptides >3 kDa, such as intact proteins, non-tryptic peptides, and/or large endogenous peptides. 13 Although proteomics has traditionally been a field ripe for automation, 14 data acquisition solutions for MS/MS of proteins or peptides at >50 000 resolving power remain relatively underdeveloped.In a typical bottom-up LC-MS/MS experiment using the new breed of ion trap-Fourier transform (FT) hybrid instruments, intact peptide data are now routinely acquired at FTMS resolution, 15,16 substantially clarifying protein identifications by database retrieval. However, parameters that lead to increased MS/MS data quality (e.g., long ion accumulation times, detection by FTMS, and spectral averaging) are sacrificed to increase the speed of MS/MS sampling in order to maximize proteome coverage. This increase in sampling rate is not

A sequential reaction methodology is employed for the complete derivatization of protein thiols, amines, and acids in high purity under denaturing conditions. Following standard thiol alkylation, protein amines are modified via reductive methylation with formaldehyde and pyridine-borane. Protein acids are subsequently amidated under buffered conditions in DMSO using the coupling reagent (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate. The generality of the approach is demonstrated with four proteins and with several amines yielding near-quantitative transformations as characterized by high-resolution Fourier transform mass spectrometry. The developed approach has numerous implications for protein characterization and general protein chemistry. Applications in mass spectrometry (MS) based proteomics of intact proteins (top-down MS) are explored, including the addition of stable isotopes for relative quantitation and protein identification through functional group counting. The methodology can be used for altering the physical and chemical properties of proteins, as demonstrated with amidation to modify protein isoelectric point and through derivatization with quaternary amines. Additionally, the chemistry has applications in the semisynthesis of monodisperse polymers based on protein scaffolds. We prepare proteins modified with azides and alkynes to enable further functionalization via copper(I)-catalyzed 1,3-dipolar Huisgen cycloaddition ("click") chemistry.